Project Summary: Mitochondria are essential to life, as they not only produce the energy our cells need to maintain cellular homeostasis but are also involved in cellular signaling and immunity. Mitochondrial dysfunction has been implicated in the pathogenesis of various diseases. In addition, mitochondrial DNA (mtDNA), which can escape the mitochondria in circumstances that are still being elucidated, can activate cytoplasmic DNA sensors to trigger an immune response. Circulating mtDNA increases with age, indicating that release of mtDNA from the mitochondria and the subsequent activation of immune signaling may be a physiologically relevant process that may contribute to the development of age-related pathology. Our lab discovered that decreased levels of the mitochondrial protein Transcription Factor A, mitochondrial (TFAM), which functions in packaging mtDNA inside the mitochondria, leads to enhanced release of mtDNA into the cytoplasm. TFAM protein levels have been shown to decrease with age in various animal models, but the consequences of increased cytoplasmic mtDNA on the aging process has not been explored. Our lab has developed a Tfam heterozygous knock-out (Tfam+/-) mouse model of enhanced mtDNA release. Cells and tissues from these mice show no significant differences in oxygen consumption rates or mtDNA encoded transcripts when compared to wild-type (WT) mice despite having a 50% depletion of mtDNA and enhanced mtDNA release into the cytoplasm. Mouse embryonic fibroblasts and bone-marrow derived macrophages will be generated from WT and Tfam+/- mice to test the activation of autophagy in response to cytoplasmic mtDNA. Since autophagy proteins have been shown to regulate the activity of the cGAS-STING pathway when bacterial DNA acts as a trigger, the dependency of both cGAS activation, STING activation, and the presence of various autophagy proteins will be tested to fully elucidate the mechanism by which autophagy is activated in this context. Activation of cGAS is a key process in the activation of the senescence program. Cellular senescence underlies many age-related pathologies. Mouse primary lung fibroblasts will be generated from WT and Tfam+/- mice to test the effect of mtDNA-mediated signaling on the rate of onset of senescence. In addition, the onset of senescence in vivo and the contribution of mtDNA-mediated signaling to the development of age-related pathology will be investigated using a well-established frailty scale. This project will shed light on the mechanisms by which mtDNA-mediated signaling is regulated, and the contribution of mtDNA pro-inflammatory signaling to the progression of age-related pathology. Understanding the molecular biology underlying the aging-process will help identify novel therapeutic interventions to extend healthy lifespan.